Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes such as cytokinesis. This laboratory has isolated two different cDNAs for nonmuscle myosin heavy chains (MHCs), MHC-A and MHC-B, which are encoded by two different genes in both human and chickens. We have demonstrated tissue and cell type-dependent expression for the two MHC mRNAs as well as changes in mRNA expression associated with cell growth and differentiation. Additional neuron- specific forms of MHC-B, which may be generated by alternative splicing of RNA, have also been demonstrated. To understand the mechanism responsible for regulating the expression of the nonmuscle MHC genes, we have isolated genomic clones which encode the promoter and flanking region for human nonmuscle MHC-B by screening human genomic DNA libraries using the 5' end fragments of nonmuscle MHC-B cDNA. Five overlapping clones were isolated and partially characterized by restriction mapping and sequencing. The clones spanned approximately 35 kbp of genomic DNA which include the first and second exons. The results of primer extension using RNA obtained from human leukemia (Jurkat) and neuroblastoma (SK-N-SH) cells suggest that there are multiple transcription start sites. The first exon consists of 71 nt or 68 nt of untranslated sequence. The second exon contains 31 nt of 5' untranslated sequence followed by 345 nt of coding sequence including the initiating ATG codon. The first intron is approximately 7 kbp in size and the size of the second intron is over 10 kbp. Sequence analysis of about a 1 kbp upstream from the first exon showed that there is no TATA box and that the GC content is high around the transcription start sites. This is consistent with the nonmuscle MHC-B gene belonging to a family of housekeeping genes. Those properties resemble that of the MHC-A gene which we reported previously. Identification of the promoter region of this gene is in progress.